Method of preparing delta9(11)-estrone

ABSTRACT

$9(11)-ESTROME ISPREPARED FROM 19-HYDROXY CHOLESTEROL BY FERMENTATION USING A CORYNEBACTERIUM SPECIES.

United States Patent Ofice. .milifail.

3 741 870 TABLE I-Continued METHOD OF PREPAR ING A -ESTRNE Colony characteristics on: Characteristics of A240l4 Gordon E. Mallett, Woking, England, assignor to Eli Trypticase soy ajar Circular, raised, reddish Lilly and Company, Indianapolis, Ind. brown colonies with mar- No Drawing. Filed Oct. 12, 1971, Ser. No. 188,547 5 i entire" In C C c 7/ 167/18 Czapeks agar Circular, raised, white colo UcS- Cl- F 1 Claim nies entire margin Sabourands glucose Very scant growth. ABSTRACT OF THE DISCLOSURE Tomato-paste oatmeal No growth.

' 10 Nutrient agar Growth sparse; circular M -estrone is prepared from 19-hydroxy cholesterol by fermentation using a Corynebacterium species. Slightly umbonate pale yellow colonies with margins entire. BACKGROUND OF THE INVENTION Carbohydrate a I a Sih et al., J. Am. Chem. Soc. 87, 2765 (1965) have femematm fif f 13 15 2352125; described the conversion of 1-9-hydroxycholesterol to 6056 lactose mannitol estrone using Nocardia species such as Nocardia restrictus sucrgse or maltose in a submerged culture fermentation.

AwnEstmhe and related compounds have been P This invention is further illustrated by the following pared according to US. Pat. 3,258,471, by the treatment ifi examples; of i10p-carboxy-A -estrene-3,17-dione with iodine, followed by a period of heating. A mixture of estrone and EXAMPLE I M -estrone is obtained. Hydrogenation of the A estrone thus produced yields estrone itself. C A g i Afi g l' l 1 e g t d f oryne ac erium sp. e s an was men a e or SUMMARY OF THE INVENTION about 3 days at C. The culture of A24014 thus pro- This invention provides a th d f preparing 01)- duced was used to inoculate Trypticase soy broth medium estrone from 19-hydroxycholesterol 3-acetate by fermencontained in shake fiasks- These flasks Were Shaken 011 a ratio with a Corynebacterium ,-NRRL 13-3931, b- 30 rotary shaker for 3 days at 30 C. Five ml. of the vegetatainable from the ARS Cult Coll i N h tive growth thus produced was used to inoculate each of Regional Research Laboratories, Peoria, 111., to be desig- 20 flasks containing 100 1111- Of Trypticase Y broth nated hereinafter as A24014. In practicing the invention, medium- These flasks were shaken 011 a rotary shaker for.

a culture of Corynebacterium A24014 is employed to 2 y at At this Point 2 of an acetone sohlinoculate a tank containing a suitable growth medium tion containing 50 of l9-hydroxycholesterol Bacetate such as Trypticase soy broth and, after an initial growth was added to each flask and the incubation continued for period, usually b t t d th 19.1 d h 1 1 another 3 days. The flask contents were combined and the substrate in solution in acetone or other suitable solvent combined broths were extracted twice with 1 of methyli dded t the f r t ti di enechloride. The methylenechloride extracts were washed The organism employed in the practice f thi i with water and dried. Evaporation of the solvent yielded tion, A24014, has the following cultural characteristics a residue which was dissolved in 5 Percent acetone in as determined by the procedures outlined in Bergys Pehtahe and chromatographed Over 50 0f fiofisil- The Manual of Determihafive Bacteriology, 7th edition (R. S, chromatogram was developed with successive 1 1. portions Breed, E, G, 1). Murray, 13 N, s i eds William of pentane containing increasin quantities of acetone. 0. Wilkins, 1953, Baltimore, Md.). All studi were Fractions containing 10 percent acetone in pentane up to ducted at 30 C. unless otherwise noted. Morphology and 20 P aeetohe ih Pehtahe were Pooled and the motility were observed on Trypticase soy agar and broth Vents removed y evaporation The ultraviolet spectrum from 18 to 72 hour in b ti ti C b i i of the residue thus obtained was consistent with that to cultural characteristics and physiological studies were obhe expected from Mun-estrone, indicating that served t 7 days estrone was produced during the fermentation.

TABLE I Property observed: Characteristics of A24014 EXAMPLE H Morphology Cells 0.72.1u by 0.7-1.4;t; A lyophilized pallet of Corynebacterium sp. A24014 r ngi g from c oid to was used to inoculate modified Trypticase soy broth regularly shaped cells, medium. The inoculated culture was allowed to grow oeeul'ihg singly i11 P for two days at 30 C. on a rotary shaker. Fifty ml. of

s pe swellthe vegetative growth thus produced was used to inoculhgs or rudimentary late 1000 ml. of the same media and the fermentation branching is observed in allowed to proceed for 2 days at 30 C. on a rotary liqu d media. shaker. This second stage vegetative cultuer was used to Motility Negat ve. inoculate 25 1. of modified Trypticase soy broth con- Acid-fastness Negatlv tained in a 44 l. fermentor with .25 l. of an anti-foam Gram-stain Posltive. agent added. After 2 days incubation, 4.75 g. of 19- Lltmus In 1lk N0 f hydroxycholesterol 3-acetate in acetone was added and Gelatm hqllefiachon y the fermentation continued for another 2 days. The ste- Oxygen requirements Aerob c. roidal material was extracted as before with methylene- Indole production Negative. chloride and the solvent evaporated to yield a dry ex- H S Production Negative. tract. The extract was dissolved in l l. of 0.1 N aqueous Temperature sodium hydroxideand ml. of methanol. The alkaline requirements Growth from 20 to 37; solution was then extracted with 1.5 l. of pentane. The

no growth at 43. aqueous and organic layers were separated, the aqueous layer extracted with pentane and the pentane layer extracted with the same mixture of aqueous sodium hydroxide and methanol as before. The pentane layers were combined and 3.1 g. of 19-hydroxycholesterol acetate starting material obtained therefrom by evaporation of the solvents. The aqueous layers were combined and acidified to pH=3-4. A -Estrone, which was insoluble in the acidic medium, was extracted with methylenechloride. Evaporation of the methylene chloride yielded 1.42 g. of a residue comprising A -estmne which was purified by chromatography over florisil using chloroform as the eluant. Taking 50 ml. fractions, fractions 3-6 produced 60 mg. of crystalline A -estrone.

In the above fermentation, Trypticase soy broth modified contains the following ingerdients per liter.

Trypticase soy medium 30 Peptone 10 Glucose 3.75

Water to 1 l.

.7 In carryingrout the above fermentations 1'9.-hydroxycholesterol 3-acetate with Corynebacterium sp. A24014 in a submerged culture fermentation under aerobic conditions to produce A -estrone and then isolating said A -estrone. h

References Cited UNITED STATES PATENTS 3,398,054 8/1968 Vezina et a1. 195 51 G 3,507,749 4/1970 Sih 1 95 51 G ALVIN E. TANENHOLTZ, Primary Examiner US. Cl. X.R. 195-51 G I 

